杜仲叶乙醇提取物的降糖作用机理

为了阐明杜仲叶的降血糖功效及其机制，采用酶-抑制剂模型，筛选高抑制率的杜仲叶乙醇提取物并判断其对α-葡萄糖苷酶的抑制类型；借助Caco-2人结肠腺癌细胞模型，研究杜仲叶20%乙醇解吸物对α-葡萄糖苷酶活性及葡萄糖转运蛋白的影响；气相色谱-质谱联用分析杜仲叶20%乙醇解吸物主要成分。杜仲叶20%乙醇解吸物可竞争性地抑制α-葡萄糖苷酶，其抑制常数Ki值为32.90 mg/mL。它对Caco-2细胞中的α-葡萄糖苷酶有较强抑制作用，其IC50为0.57 mg/mL，且对Caco-2细胞摄取葡萄糖也有一定抑制作用，1 mg/mL时抑制率可达26.25%。气相色谱-质谱分析表明杜仲叶20%乙醇解吸物主要有DL-异柠檬酸内酯、百里酚、儿茶素、2,6-二羟基-苯甲酸和3,4-二羟基-苯丙烯酸等。这些结果表明，杜仲叶乙醇提取物可以通过抑制α-葡萄糖苷酶活性，降低葡萄糖吸收来实现降血糖的效果。

Ethanol Extract of Eucommia ulmoides Leaves Inhibits Alpha-Glucosidase in Caco-2 Cells

The inhibitory effects of 40% ethanol extract of Eucommia ulmoides leaves on alpha-glucosidase and glucose
transport were investigated in Caco-2 cells in vitro to elucidate the anti-hyperglycemic mechanism of Eucommia ulmoides
leaves. The alpha-glucosidase inhibitors in the ethanol extract were also identified by GC-MS analysis. The kinetic study
showed that the 20% ethanol elution fraction (EEB) from macroporous resin AB-8 of the ethanol extract of Eucommia
ulmoides leaves could competitively inhibit alpha-glucosidase with a Ki value of 32.90 mg/mL, indicating that alphaglucosidase
has a strong affinity with the extract. Meanwhile, the inhibition was found to be reinforced as the concentration
of EEB increased. For investigation of the inhibitory effect of EEB in Caco-2 cells, cytotoxicity assays were firstly conducted
by using different concentrations of EEB (0.05, 0.10, 0.20, 0.50 and 1.00 mg/mL) to determine the safety level at which cell
growth and viability could not be affected. All tested concentrations of EEB had no effect on the growth or viability of Caco-2
cells. Subsequently, EEB at various concentration exhibited effective suppression on alpha-glucosidase by using maltose
(28 mmol/L) as the substrate in Caco-2 cells in a concentration-dependent manner with an IC50 of 0.57 mg/mL. Furthermore,
EEB could also attenuate glucose transport in Caco-2 cells, which could be decreased to 26.25% by EEB (1 mg/mL). These
results indicate that EEB exerts strong anti-hyperglycemic effect by suppressing disaccharidase and glucose transportors.
GC-MS analysis was conducted to elucidate the composition of the ethanol extract of Eucommia ulmoides leaves. It was
found that acids, monosaccharides, polyphenols, esters, amino acids were the main components of EEB. Further analysis by
GC-MS revealed that the ethanol extract was rich in DL-isocitric acid lactone, thymol, catechin, 2,6-dihydroxybenzoic acid
and 3,4-dihydroxy cinnamic acid. These compounds may play important roles in the hypoglycemic effect of the extract. The
present study suggests Eucommia ulmoides leaves are a medicinal food material for preventing and treating diabetes, and
could be further developed into healthful products.